Sequences Diagnostic For Shrimp Pathogens

ABSTRACT

Primers have been isolated that are diagnostic for the detection of the white spot syndrome virus (WSSV). The primers are based on a new portion of the WSSV genome and may be used in primer directed amplification or nucleic acid hybridization assay methods.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority under 35 U.S.C. §119 from U.S.Provisional Application Ser. No. 60/839,744, filed Aug. 24, 2006.

FIELD OF THE INVENTION

The invention relates to the field of diagnostic testing. Morespecifically, new primers have been developed for use in detection ofthe White Spot Syndrome Virus pathogen of shrimp.

BACKGROUND OF THE INVENTION

Commercial shrimp farms suffer extensive losses due to the effects of anumber of common pathogens. White Spot Syndrome Virus (WSSV) can causerapid death in the commercial shrimp, Penaeus monodon. WSSV is a doublestranded DNA baculovirus, the complete genome of which has beensequenced (van Hulten et al. Virology 286:7-22 (2001); and Yang et al.J. Virol. 75:11811-11820 (2001)). There are at least 12 variants of WSSVfound in Thailand that are distinguished by differences in multiplerepeat lengths in open reading frame (ORF) 94.

WSSV spreads rapidly and can devastate a commercial shrimp operationwithin two weeks. Detection of WSSV in hatchery broodstock and inpost-larvae allows infected shrimp to be eliminated before entry into acommercial production system. Consequently, a variety of methods havebeen developed for the detection of WSSV in shrimp, including nucleicacid-based methods and immunological methods (You et al., Current Topicsin Virology 4:63-73 (2004); and Lightner et al., Aquaculture164(1):201-220 (1998)). Polymerase chain reaction (PCR) methods are ofparticular interest because they are simple, rapid, and sensitive. PCRmethods for the detection of WSSV, which are based on amplifyingdifferent diagnostic regions of the genome, have been described (see forexample, Kou et al., U.S. Pat. No. 6,190,862; Lee, U.S. Pat. No.6,872,532; Hameed et al., Aquaculture International 13(5):441-450(2005); Jian et al., Diseases of Aquatic Organisms 67(1&2):171-176(2005); and Durand et al., Journal of Fish Diseases 25(7):381-389(2002)). Additionally, a PCR-based method, specifically the WSSV-232assay, has been used in the shrimp industry in Thailand. The assayinvolves detection of WSSV DNA using primers for a target sequence inORF 21 (Kiatpathomchai et al., J. of Virology Methods 130:79-82 (2005)).However, there have been outbreaks of WSSV infection following testingfor WSSV using the WSSV-232 assay which have been attributed toinsufficient sensitivity (Kiatpathomchai et al., supra). Clearly, newand more sensitive assays for the detection of WSSV are needed.

All of the above methods are useful for the detection of WSSV; however,they generally suffer from a lack of specificity, sensitivity, or arecomplex and time consuming. Additionally, because of the high genemutation rate in the virus, tests directed to different regions of thegenome would be useful. Therefore, there is a need for a highlysensitive assay for WSSV that is rapid, accurate and easily used in thefield. The stated problem is addressed herein by the discovery ofprimers based on new portions of the WSSV genome. The primers identifiedherein can be used in primer directed amplification or nucleic acidhybridization assay methods for the detection of WSSV without theproblems associated with previous methodologies.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides an isolated WSSVdiagnostic primer sequence as set forth in any one of SEQ ID NOs:1-8 oran isolated nucleic acid molecule that is completely complementary toSEQ ID NOs:1-8.

In another embodiment, the invention provides a pair of two differentWSSV diagnostic primer sequences as disclosed herein, wherein the pairis capable of priming a nucleic acid amplification reaction thatamplifies a region of nucleic acid within the WSSV genome.

In another embodiment, the invention provides a kit for the detection ofWSSV comprising at least one pair of WSSV diagnostic primer sequencesdisclosed herein.

In another embodiment, the invention provides a method for detecting thepresence of WSSV in a sample comprising:

-   -   (i) providing DNA from a sample suspected of containing the        WSSV; and    -   (ii) probing the DNA with a probe derived from the isolated WSSV        diagnostic primer sequence of any of SEQ ID NOs:1-8 under        suitable hybridization conditions;

wherein the identification of a hybridizable nucleic acid fragmentconfirms the presence of WSSV.

In other embodiments the detection methods identify DNA samples that arenot infected with WSSV.

In another embodiment, the invention provides a method for detecting thepresence of WSSV in a sample comprising:

-   -   (i) providing DNA from a sample suspected of containing WSSV;        and    -   (ii) amplifying the DNA with at least one pair of WSSV        diagnostic primer sequences disclosed herein such that        amplification products are generated;    -   wherein the presence of amplification products confirms the        presence of WSSV.

In another embodiment, the invention provides a method for quantifyingthe amount of WSSV in a sample comprising:

-   -   (i) providing DNA from a sample suspected of containing WSSV;

(ii) amplifying the DNA with at least one pair of WSSV diagnostic primersequences disclosed herein by thermal cycling between at least adenaturing temperature and an extension temperature in the presence of anucleic acid-binding fluorescent agent or a fluorescently labeled probe;

-   -   (iii) measuring the amount of fluorescence generated by the        nucleic acid-binding fluorescent agent or the fluorescently        labeled probe during the thermal cycling;    -   (iv) determining a cycle threshold number at which the amount of        fluorescence generated by the nucleic acid-binding fluorescent        molecule or the fluorescently labeled probe reaches a fixed        threshold value above a baseline value; and    -   (v) calculating the amount of WSSV in the sample by comparing        the cycle threshold number determined for the WSSV in the sample        with a standard curve of the cycle threshold number versus the        logarithm of template concentration determined using standard        solutions of known concentration.

BRIEF DESCRIPTION OF THE FIGURE AND SEQUENCE DESCRIPTIONS

The various embodiments of the invention can be more fully understoodfrom the following detailed description, figure, and the accompanyingsequence descriptions, which form a part of this application.

FIG. 1A shows the melting curve for the WSSV product and the actininternal sample control product formed by simultaneous PCR amplificationof the WSSV virus DNA and actin DNA, as described in Example 10. Themelting temperature (Tm) values of the WSSV and actin products areindicated on their corresponding melting curves.

FIG. 1B shows the results of the agarose gel electrophoresis separationof samples containing the WSSV product and the actin internal samplecontrol product formed by simultaneous PCR amplification of the WSSVvirus DNA and actin DNA, as described in Example 10. The quantity ofWSSV and shrimp DNA is shown above each lane; “M” is a 100-bp DNAladder.

The following sequences conform with 37 C.F.R. 1.821-1.825(“Requirements for patent applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) and areconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (1998) and the sequence listing requirements of the EPO and PCT(Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of theAdministrative Instructions). The symbols and format used for nucleotideand amino acid sequence data comply with the rules set forth in 37C.F.R. §1.822.

SEQ ID NOs:1-8 are the nucleotide sequences of WSSV diagnostic primersuseful for detection of WSSV.

SEQ ID NOs:9-12 are the nucleotide sequences of synthetic WSSV templatesdescribed in the General Methods Section of the Examples. Thesesequences are also the nucleotide sequences of amplification productsobtained using pairs of WSSV diagnostic primers disclosed herein.

SEQ ID NOs:13-16 are the nucleotide sequences of internal sample controlprimers described in Example 10.

SEQ ID NOs:17-19 are the nucleotide sequences of the fluorescentlylabeled probes described in Examples 11 and 12.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are primers useful in assays for the detection of whitespot syndrome virus. The primers may be used in nucleic acidamplification methods as well as in hybridization assays for theefficient detection and quantification of virulent white spot syndromevirus.

In this disclosure, a number of terms and abbreviations are used.

The following definitions are provided and should be referred to forinterpretation of the claims and the specification.

“Polymerase chain reaction” is abbreviated PCR.

“White Spot Syndrome Virus” is abbreviated WSSV.

The term “isolated WSSV diagnostic primer sequence” refers to a sequencecorresponding to a portion of the WSSV genome being diagnostic for thepresence of WSSV.

As used herein, an “isolated nucleic acid fragment” is a polymer of RNAor DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. An isolated nucleicacid fragment in the form of a polymer of DNA may be comprised of one ormore segments of cDNA, genomic DNA or synthetic DNA.

The term “amplification product” or “amplicon” refers to the nucleicacid fragment that is produced during a primer directed amplificationreaction. Typical methods of primer directed amplification includepolymerase chain reaction (PCR), ligase chain reaction (LCR), stranddisplacement amplification (SDA), or other isothermal amplificationprocesses. If PCR methodology is selected, the replication compositionwould typically include, for example: deoxynucleotide triphosphates, twoprimers with appropriate sequences, a thermostable DNA polymerase andproteins. These reagents and details describing procedures for their usein amplifying nucleic acids are provided in U.S. Pat. No. 4,683,202(1987, Mullis, et al.) and U.S. Pat. No. 4,683,195 (1986, Mullis, etal.). If LCR methodology is selected, then the nucleic acid replicationcompositions would comprise, for example: a thermostable ligase (e.g.,T. aquaticus ligase), two sets of adjacent oligonucleotides (wherein onemember of each set is complementary to each of the target strands),Tris-HCl buffer, KCl, EDTA, NAD, dithiothreitol and salmon sperm DNA(see for example, Tabor et al., Proc. Natl. Acad. Sci. U.S.A.,82:1074-1078 (1985)).

The term “primer” refers to an oligonucleotide (synthetic or occurringnaturally), which is capable of acting as a point of initiation ofnucleic acid synthesis or replication along a complementary strand whenplaced under conditions in which synthesis of a complementary stand iscatalyzed by a polymerase.

The term “thermal cycling” refers to the entire pattern of changingtemperature used during certain nucleic acid amplification methods, suchas PCR and LCR. This process is common and well known in the art. See,for example, Sambrook, J., Fritsch, E. F. and Maniatis, T., MolecularCloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor LaboratoryPress: Cold Spring Harbor, N.Y. (1989); and U.S. Pat. No. 4,683,202 toMullis et al. and U.S. Pat. No. 4,683,195 to Mullis et al. In general,PCR thermal cycling includes an initial denaturing step at hightemperature, followed by a repetitive series of temperature cyclesdesigned to allow template denaturation, primer annealing, and extensionof the annealed primers by the polymerase.

The term “cycle threshold number”, also referred to herein as “CT”,refers to the cycle number during thermal cycling at which the amount offluorescence due to product formation reaches a fixed threshold valueabove a baseline value.

The term “probe” refers to an oligonucleotide (synthetic or occurringnaturally) that is significantly complementary to a target sequence,also referred to herein as a “fragment”, (i.e., the sequence to bedetected or a portion of the sequence to be detected) and forms aduplexed structure by hybridization with at least one strand of thetarget sequence. The probe can be labeled to facilitate detection, forexample, using a fluorescent label or a ligand label.

The term “replication inhibitor moiety” refers to any atom, molecule orchemical group that is attached to the 3′ terminal hydroxyl group of anoligonucleotide that will block the initiation of chain extension forreplication of a nucleic acid strand. Examples include, but are notlimited to, 3′ deoxynucleotides (e.g., cordycepin), dideoxynucleotides,phosphate, ligands (e.g., biotin and dinitrophenol), reporter molecules(e.g., fluorescein and rhodamine), carbon chains (e.g., propanol), amismatched nucleotide or polynucleotide, or peptide nucleic acid units.

The term “non-participatory” refers to the lack of participation of aprobe or primer in a reaction for the amplification of a nucleic acidmolecule. Specifically, a non-participatory probe or primer is one thatwill not serve as a substrate for, or be extended by, a DNA polymerase.A “non-participatory probe” is inherently incapable of being chainextended by a polymerase. It may or may not have a replication inhibitormoiety.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under suitable conditions of temperature and solution ionicstrength. Hybridization and washing conditions are well known andexemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. MolecularCloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor LaboratoryPress: Cold Spring Harbor, N.Y. (1989), particularly Chapter 11 andTable 11.1 therein (entirely incorporated herein by reference). Theconditions of temperature and ionic strength determine the “stringency”of the hybridization. For preliminary screening for homologous nucleicacids, low stringency hybridization conditions, corresponding to amelting temperature (Tm) of 55° C., can be used, e.g., 5×SSC, 0.1% SDS,0.25% milk, and no formamide; or 30% formamide, 5×SSC, 0.5% SDS.Moderate stringency hybridization conditions correspond to a higher Tm,e.g., 40% formamide, with 5× or 6×SSC. Hybridization requires that thetwo nucleic acids contain complementary sequences, although depending onthe stringency of the hybridization, mismatches between bases arepossible. The appropriate stringency for hybridizing nucleic acidsdepends on the length of the nucleic acids and the degree ofcomplementation, variables well known in the art. The greater the degreeof similarity or homology between two nucleotide sequences, the greaterthe value of Tm for hybrids of nucleic acids having those sequences. Therelative stability (corresponding to higher Tm) of nucleic acidhybridizations decreases in the following order: RNA:RNA, DNA:RNA,DNA:DNA. For hybrids of greater than 100 nucleotides in length,equations for calculating Tm have been derived (see Sambrook et al.,supra, 9.50-9.51). For hybridizations with shorter nucleic acids, i.e.,oligonucleotides, the position of mismatches becomes more important, andthe length of the oligonucleotide determines its specificity (seeSambrook et al., supra, 11.7-11.8). In one embodiment, the length for ahybridizable nucleic acid is at least about 10 nucleotides. Preferably,a minimum length for a hybridizable nucleic acid is at least about 15nucleotides; more preferably at least about 20 nucleotides; and mostpreferably the length is at least 30 nucleotides. Furthermore, theskilled artisan will recognize that the temperature and wash solutionsalt concentration may be adjusted as necessary according to factorssuch as length of the probe.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers to any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of effecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide.

The terms “plasmid”, “vector” and “cassette” refer to an extrachromosomal element often carrying genes which are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA molecules. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitate transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include, but is not limited to, the GCG suite of programs(Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison,Wis.), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.215:403-410 (1990), DNASTAR (DNASTAR, Inc., Madison, Wis.), and VectorNTI® software version 7.0. Within the context of this application itwill be understood that where sequence analysis software is used foranalysis, that the results of the analysis will be based on the “defaultvalues” of the program referenced, unless otherwise specified. As usedherein “default values” will mean any set of values or parameters whichoriginally load with the software when first initialized.

Standard recombinant DNA and molecular cloning techniques used here arewell known in the art and are described by Sambrook, J., Fritsch, E. F.and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2^(nd) ed.,Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y. (1989)(hereinafter “Maniatis”); and by Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, published by Greene Publishing Assoc.and Wiley-Interscience (1987).

White Spot Syndrome Virus Genome

The white spot syndrome virus (WSSV), also known as the white spotbacilliform virus (WSBV), is a major shrimp pathogen with a highmortality rate and a wide host range. The complete genome of WSSV hasbeen sequenced (van Hulten et al., Virology 286:7-22 (2001); and Yang etal., J. Virol. 75:11811-11820 (2001)). The genome consists of doublestranded, circular DNA containing 305, 107 base pairs (bp) and 181 openreading frames (ORFs) (GenBank AF332093). There are at least 12 variantsof WSSV found in Thailand that are distinguished by differences inmultiple repeat lengths in ORF 94 (Wongteerasupaya et al., Dis. Aquat.Org. 54:253-257 (2003)).

WSSV Diagnostic Primer Sequences

Disclosed herein are diagnostic primer sequences useful in a variety ofassay formats for high sensitive detection of WSSV. These primers aredirected to regions of the WSSV genome not previously used for WSSVdetection.

Primer sequences were empirically identified using a series of “insilica” (i.e. computer-based) sequence analysis tools. In this process,a database was assembled containing all known WSSV sequences. Thesesequences were first aligned and then analyzed for primer sites usingVector NTI® software (InforMax Inc., Bethesda, Md.) based on homologywith other WSSV sequences, a specified amplicon length, saltconcentration, Tm (melting temperature), C+G content and freedom fromhairpin and secondary structure parameters. Prospective primers werethen screened against GenBank sequences. Those primers established tocontain less than 5 bases of homology with other non-target genesequences were selected for experimental investigation of PCRamplification efficiency and minimal primer-dimer formation. Primersshowing both high amplification efficiency and minimal primer-dimerformation were selected for testing with a panel of DNA isolated fromshrimp infected with various shrimp pathogens and DNA from shrimpcertified to be disease free. Those primers amplifying all WSSV strainsand showing no response to both DNA from shrimp infected with non-WSSVpathogens and to DNA isolated from different species of certifieddisease-free shrimp were selected as useful primers.

The primer sequences found to be useful in the detection of WSSV andtheir location in the WSSV genome are given in Table 1. These primersmay be synthesized using standard phosphoramidite chemistry or may bepurchased from companies such as Sigma Genosys (The Woodlands, Tex.).TABLE 1 WSSV Diagnostic Primer Sequences WSSV Genome SEQ ID LocationPrimer, Direction NO: ORF (GenBank AF332093) WSSV77F, Forward 1 7761335-61358 WSSV77R, Reverse 2 77 61420-61443 WSSV54F, Forward 3 5431287-31309 WSSV54R, Reverse 4 54 31391-31414 WSSV56F, Forward 5 5633145-33168 WSSV56R, Reverse 6 56 33269-33292 WSSV130F, Forward 7 130146110-146132 WSSV130R, Reverse 8 130 146212-146234Assay Methods

The primer sequences disclosed herein may be used in a variety of assayformats for the detection and quantification of WSSV. The two mostconvenient formats rely on methods of nucleic acid hybridization orprimer directed amplification methods such as PCR.

Primer Directed Amplification Assay Methods

In one embodiment, the present WSSV diagnostic primer sequences may beused in primer directed nucleic acid amplification for the detection ofthe presence of WSSV. A variety of primer directed nucleic acidamplification methods are well known in the art and are suitable for usewith the primers disclosed herein. These nucleic acid amplificationmethods include thermal cycling methods (e.g., polymerase chain reaction(PCR) and ligase chain reaction (LCR)), as well as isothermal methodsand strand displacement amplification (SDA).

LCR methods are well known in the art (see for example, Tabor et al.,Proc. Natl. Acad. Sci. U.S.A., 82:1074-1078 (1985)). Typically, LCRnucleic acid replication compositions comprise, for example: athermostable ligase (e.g., T. aquaticus ligase), two sets of adjacentoligonucleotide primers (wherein one member of each set is complementaryto each of the target strands), Tris-HCl buffer, KCl, EDTA, NAD,dithiothreitol and salmon sperm DNA.

SDA methods are also well known in the art. An in depth discussion ofSDA methodology is given by Walker et al. (Proc. Natl. Acad. Sci.U.S.A., 89:392 (1992)). Typically in SDA, two oligonucleotide primersare used, each having regions complementary to only one of the stands inthe target. After heat denaturation, the single-stranded targetfragments bind to the respective primers which are present in excess.Both primers contain asymmetric restriction enzyme recognition sequenceslocated 5′ to the target binding sequences. Each primer-target complexcycles through nicking and polymerization/displacement steps in thepresence of a restriction enzyme, a DNA polymerase and threedeoxynucleotide triphosphates (dNTPs) and one deoxynucleotide α-thiotriphosphate (dNTP[aS]).

The preferred method for detecting WSSV using the diagnostic primersequences disclosed herein is PCR, which is described by Mullis et al.in U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,683,195, which are bothspecifically incorporated herein by reference. In PCR methods, the WSSVdiagnostic primer sequences disclosed herein and their completecomplimentary sequences are used in pairs which are capable of priming anucleic acid amplification reaction that amplifies a region within theWSSV genome. Various combinations of the WSSV diagnostic primersequences and their compliments can be used. Suitable primer pairsinclude, but are not limited to, SEQ ID NOs:1 and 2, SEQ ID NOs:3 and 4,SEQ ID NOs:5 and 6, SEQ ID NOs:7 and 8, SEQ ID NOs:3 and 6, SEQ ID NO:3and the complete complement of SEQ ID NO:5, and SEQ ID NO:6 and thecomplete compliment of SEQ ID NO:4. Generally, the two primers are mixedwith the sample DNA, a mixture of four deoxynucleotide triphosphates(i.e., dATP, dCTP, dTTP, and dGTP), a thermostable DNA polymerase, suchas Taq DNA polymerase, in a buffer solution. This mixture is thenthermal cylcled using a thermal cycler instrument to amplify the desiredtarget region. Thermal cyclers are commercially available from manysources (e.g., Applied Biosystems (Foster City, Calif.); Brinkmann(Westbury, N.Y.); MJ Research (Waltham, Mass.); and Stratagene (LaJolla, Calif.)).

In general, PCR thermal cycling includes an initial denaturing step athigh temperature, followed by a repetitive series of temperature cyclesdesigned to allow template denaturation, primer annealing, and extensionof the annealed primers by the polymerase. Generally, the samples areheated initially for about 2 to 10 minutes at a temperature of about 95°C. to denature the double stranded DNA sample. Then, in the beginning ofeach cycle, the samples are denatured for about 10 to 60 seconds,depending on the samples and the type of instrument used. Afterdenaturing, the primers are allowed to anneal to the target DNA at alower temperature, from about 40° C. to about 60° C. for about 20 to 60seconds. Extension of the primers by the polymerase is often carried outat a temperature ranging from about 60° C. to about 72° C. The amount oftime used for extension will depend on the size of the amplicon and thetype of enzymes used for amplification and is readily determined byroutine experimentation. Additionally, the annealing step can becombined with the extension step, resulting in a two step cycling.Thermal cycling may also include additional temperature shifts in PCRassays. The number of cycles used in the assay depends on many factors,including the primers used, the amount of sample DNA present, and thethermal cycling conditions. The number of cycles to be used in any assaymay be readily determined by one skilled in the art using routineexperimentation. Optionally, a final extension step may be added afterthe completion of thermal cycling to ensure synthesis of allamplification products.

Following amplification, the amplified nucleotide sequence may beligated to a suitable vector followed by transformation of a suitablehost organism with said vector. One thereby ensures a more readilyavailable supply of the amplified sequence. Alternatively, followingamplification, the amplified sequence or a portion thereof may bechemically synthesized for use as a nucleotide probe for use in ahybridization assay, as described below. In either situation the DNAsequence of the variable region may be established using methods such asthe dideoxy method (Sanger, F. et al. Proc. Natl. Acad. Sci.74:5463-5467 (1977)). The sequence obtained is used to guide the choiceof the probe for the organism and the most appropriate sequence(s)is/are selected.

In order to detect the presence of WSSV in a sample suspected ofcontaining WSSV (e.g., shrimp or other crustaceans) using a primerdirected nucleic acid amplification method, DNA from the sample must beprovided in a form that is capable of being amplified. Typically, theDNA must be free from the cell and sample materials and may be treatedto eliminate proteins and other cell components. The DNA may be obtainedfrom any suitable tissue, fluid or sample material including, but notlimited to, shrimp tissue (e.g., gills, pleopods, hemolymph, muscle,tail, eyestalk, stomach, leg, and connective tissue), wash fluids, andpond water samples. The samples may be suspected of containing WSSV forany number of reasons, including proximity to a known contaminant orotherwise, or may only be suspected of contamination by virtue of WSSV'scommon presence in the commercial shrimp industry. Thus, a samplesuspected of containing WSSV can be any DNA sample described above.

Methods for providing DNA, which is suitable for amplification, fromtissues are well known in the art. For example, DNA may be extractedfrom a sample by homogenizing the tissue or sample material in Tris-HClbuffer, centrifuging to remove solid debris, isolating the DNA bytreatment with proteinase K and sarkosyl, extracting the DNA with phenoland chloroform-isoamyl alcohol, and precipitating the DNA with absoluteethanol, as described by Kiatpathomchai et al. (J. of Virology Methods130:79-82 (2005)). Alternatively, the methods for isolating WSSV viralDNA from various tissues described by Yoganandhan et al. (AquacultureResearch 34(12):1093-1097 (2003)) and Kou et al. (U.S. Pat. No.6,190,862) may be used. Additionally, the DNA can be provided in a formwhich is suitable for amplification using a commercially available DNAisolation kit, such as the QIAamp DNA Mini Kit (Qiagen, ValenciaCalif.), the QIAamp Tissue Kit (Qiagen), or the High Pure PCR TemplatePreparation Kit (Roche Applied Science, Indianapolis, Ind.).

The DNA is then amplified with at least one pair of WSSV diagnosticprimer sequences disclosed herein using a nucleic acid amplificationmethod, as described above. A combination of different pairs ofdiagnostic primer sequences may also be used. The presence of theamplification product, detected as described below, confirms thepresence of WSSV in the sample. In one embodiment, PCR is used toamplify the DNA.

In nucleic acid amplification methods, test results can bemisinterpreted due to reagent failure, procedural errors, and instrumentmalfunction. Additionally, problems arise due to the presence ofinhibitory substances in the sample materials or degradation of thesample DNA or RNA during sample processing and nucleic acid recovery. Toovercome these problems, internal control tests can be performed incombination with the WSSV assay to alert users to these types of errorsand to aid in quantification of test results.

Two types of internal control tests can be used. One approach is basedon co-amplification of an “internal template control” (ITC), which isadded to the nucleic acid amplification reagent mixture prior toreaction. A second approach is based on co-amplification of an “internalsample control” (ISC) contained in the sample. In both cases, thesequence of the internal control DNA or RNA is different from that ofthe WSSV DNA.

The internal sample control can be a DNA or RNA gene sequence conservedor consistently present in sample materials (e.g. shrimp tissue andhemolymph). The primers used to amplify the ISC target DNA or RNA arechosen so that they do not amplify WSSV DNA and the WSSV test primersare chosen so that they do not amplify the internal sample control DNAor RNA targets. In this way, the ISC and WSSV targets amplifyindependently. In the assay, both the ISC and the WSSV targets areprocessed using the same reagents and conditions. Furthermore, bothtarget templates are amplified using the same reagents and reactionconditions. Because the ISC template and primers are present in the testsamples, ISC product should be produced during amplification. If the ISCproduct is not formed, it is an indication that the test chemistry didnot function correctly and the WSSV test results are incorrect andshould not be relied on. If the correct ISC product formation occurs, itindicates that the test chemistry worked correctly, and the WSSV sampleprocessing and test reactions are assumed to have functioned correctlyso that the WSSV test can be more accurately interpreted.

ISC primers can be selected from gene sequences of genes coding forstructural proteins, metabolic enzymes or ribosomal products of thepathogen host species which are subject to WSSV infections. For example,the ISC primers can be gene sequences derived from the shrimp actingene, or 18S, 23S or 5S ribosomal genes of shrimp, or other constitutivegenes. Suitable examples of ISC primer pairs include, but are notlimited to, SEQ ID NOs:13, 14, and SEQ ID NOs:15, 16, derived from thePenaeus monodon actin 1 gene (GenBank AF100986), as shown in Table 2.TABLE 2 Internal Sample Control (ISC) Primer Sequences Actin 1 GeneLocation Primer, Direction SEQ ID NO: (GenBank AF100986) ActinF2,Forward 13 391-411 ActinR2, Reverse 14 608-629 ActinF3, Forward 15326-346 ActinR3, Reverse 16 553-574

In one embodiment, at least one pair of ISC primers is included in thenucleic acid amplification reagent mixture in order to produce aninternal sample control product in the amplification reaction. In oneembodiment, the at least one pair of ISC primers is selected from thegroup consisting of SEQ ID NOs:13, 14, and SEQ ID NOs:15, 16.

Additionally, an internal template control (ITC) can be used toadvantage with the WSSV test primers to aid in quantification of thetest response. Primer requirements for the ITC are similar to those ofthe ISC primers with the exception that both the ITC template andprimers are added to the amplification reagent mixture. The ITC primersare chosen so that they do not amplify genomic DNA or RNA from the testspecies, such as shrimp, which are subject to WSSV. The ITC template isadded at a known concentration so that the copy number per reaction isknown. Because the ITC template is included in the amplification reagentmixture, the ITC product is produced during amplification. The amount ofITC product will vary from reaction to reaction depending on theamplification efficiency of the reaction and other variables. Sincethese same variables also affect the WSSV DNA amplification, the amountof WSSV product produced will be proportionately related to the amountof the ITC product produced in the reaction. Therefore, the copy numberof the WSSV template in the assay can be inferred from theproportionality between the ITC originally added, the ITC productformed, and the WSSV product produced. Relative product formation can bedetermined in CT units when labeled internal probes are used or by thederivative of the melting curves at the products' respective meltingtemperature.

The ITC primer sequences can be rationally designed or derived from genesequences from non-test species such as other viruses or genes fromplants and animals which are not present in the test samples. In thisway, sample materials do not contain other DNA or RNA which could beamplified by the ITC primers.

In one embodiment, at least one internal template control and at leastone pair of ITC primers are included in the nucleic acid amplificationreagent mixture in order to produce at least one ITC product in theamplification reaction.

A variety of detection methods, which are well known in the art, may beused in the methods disclosed herein. These detection methods include,but are not limited to, standard non-denaturing gel electrophoresis(e.g., acrylamide or agarose), denaturing gradient gel electrophoresis,temperature gradient gel electrophoresis, capillary electrophoresis, andfluorescence detection.

Fluorescence detection methods provide rapid and sensitive detection ofamplification products. Fluorescence detection also provides thecapability of real-time detection, wherein the formation ofamplification products is monitored during the thermal cycling process.Additionally, the amount of the initial target may be quantified usingfluorescence detection. Fluorescence detection may be done by adding anucleic acid-binding fluorescent agent to the reaction mixture eitherbefore or after the thermal cycling process. Preferably, the nucleicacid-binding fluorescent agent is an intercalating dye that is capableof non-covalent insertion between stacked base pairs in the nucleic aciddouble helix. However, non-intercalating nucleic acid-bindingfluorescent agents are also suitable.

Non-limiting examples of nucleic acid-binding fluorescent agents usefulin the methods of the invention are ethidium bromide and SYBR® Green I(available from Molecular Probes; Eugene, Oreg.). Addition of thenucleic acid-binding fluorescent agent to the reaction mixture prior tothermal cycling permits monitoring of the formation of amplificationproducts in real-time, as described by Higuchi (U.S. Pat. No.5,994,056). Thermal cyclers capable of real-time fluorescencemeasurements are commercially available from companies such as AppliedBiosystems (Foster City, Calif.), MJ Research (Waltham, Mass.), andStratagene (La Jolla, Calif.). Following amplification, confirmation ofthe amplification product can be assessed by determining the meltingtemperature of the product using methods know in the art, for example,by generating a melting curve using fluorescence measurement.

Fluorescence detection of amplification products may also beaccomplished using other methods known in the art, such as the use of afluorescently labeled probe. The probe comprises a complimentarysequence to at least a portion of the amplification product.Non-limiting examples of such probes include TaqMan® probes (AppliedBiosystems) and Molecular Beacons (Goel et al., J. Appl. Microbiol.99(3):435-442 (2005)). For example, gene sequences for the constructionof fluorescently labeled probes for use with the WSSV primers disclosedherein can be selected by analysis of the WSSV genes and test ampliconsusing commercially available analysis software such as Primer Express®v2.0 (Applied BioSystems Inc., Foster City Calif.), as described indetail in Examples 11 and 12 below. Probe sequences are selected to fallwithin the proximal ends of the specific WSSV test amplicons. Suitableprobe sequences include, but are not limited, to the sequences set forthin SEQ ID NOs:17-19. The probes may be fluorescently labeled usingmethods known in the art, such as those described below for labelinghybridization probes. For real time fluorescent detection, probes can bedual labeled. For example, the 5′ end of the probe can be labeled with afluorophore, such as 6FAM™ (Applied BioSystems), and the 3′ end can belabeled with a quencher dye, such as 6-carboxytetramethylrhodamine(TAMRA). In the case of a minor groove binding probe, the 3′ end can belabeled with a quencher dye and a minor groove binder complex.Fluorescently labeled probes may be obtained from commercial sourcessuch as Applied BioSystems.

In one embodiment, the invention provides a method for quantifying theamount of WSSV in a sample. In this embodiment, DNA is provided from asample suspected of containing WSSV, as described above. The DNA isamplified with at least one pair of the oligonucleotide primersdisclosed herein by thermal cycling between at least a denaturingtemperature and an extension temperature in the presence of a nucleicacid-binding fluorescent agent or a fluorescently labeled probe. Theamount of fluorescence generated by the nucleic acid-binding fluorescentagent or the fluorescently labeled probe is measured during thermalcycling. From the fluorescence measurements, a cycle threshold number isdetermined at which the amount of fluorescence generated by the nucleicacid-binding fluorescent agent or the fluorescently labeled probereaches a fixed threshold value above a baseline value. The cyclethreshold number is referred to herein as the CT number or value. The CTnumber can be determined manually or determined automatically by theinstrument. To determine the CT number, the baseline fluorescence isdetermined for each sample during the initial amplification cycles. Amathematical algorithm is then employed to establish what astatistically significant change in fluorescence would need to be forthe fluorescence signal to be above the background. The cycle number atwhich the florescence exceeds this threshold is referred to as the CTnumber. Typically, the more DNA present in the sample at the start ofthe thermal cycling, the fewer number of cycles it will take to reachthe threshold value. Therefore, the CT number is inversely related tothe initial amount of WSSV in the sample. After the CT number for theWSSV sample is determined, the amount of WWSV originally present in thesample can be calculated by comparing the cycle threshold numberdetermined for the WSSV in the sample with a standard curve of the cyclethreshold number versus the logarithm of template concentrationdetermined using standard solutions of known concentration, as is wellknown in the art.

Nucleic Acid Hybridization Methods

The basic components of a nucleic acid hybridization test for WSSVinclude a DNA probe, a sample suspected of containing WSSV, and aspecific hybridization method. Probes of the present invention aresingle stranded nucleic acid sequences which are complementary to thenucleic acid sequences to be detected and are “hybridizable” thereto.Typically in hybridization methods, the probe length can vary from asfew as 5 bases to several kilobases and will depend upon the specifictest to be done. Only part of the probe molecule need be complementaryto the nucleic acid sequence to be detected. In addition, thecomplementarity between the probe and the target sequence need not beperfect. Hybridization does occur between imperfectly complementarymolecules with the result that a certain fraction of the bases in thehybridized region are not paired with the proper complementary base.

The DNA probes disclosed herein are derived from the WSSV diagnosticprimer sequences described above. As used herein the phrase “derivedfrom the WSSV diagnostic primer sequences” means that the DNA probes canbe the WSSV diagnostic primer sequences, the amplification productsequences obtained therefrom using a nucleic acid amplification method,portions of the WSSV diagnostic primer sequences or the amplificationproduct sequences, or the complete complementary sequences of any of theaforementioned sequences. The term “portion”, as used above, refers toany part of the WSSV diagnostic primer sequences or the amplificationproducts obtained therefrom that is less than the complete sequence.Preferably, the length of the portion for use as a probe is at leastabout 15 bases, more preferably, at least about 20 bases. Non-limitingexamples of DNA probes derived from the WSSV diagnostic primer sequencesinclude the WSSV diagnostic primer sequences given as SEQ ID NOs:1-8,the amplification product sequences given as SEQ ID NOs:9, 10, 11, and12, and the complete complimentary sequences of SEQ ID NOs:1-12.

The probe may be labeled to facilitate detection. Methods of attachinglabels to nucleic acid probes are well known in the art. For example,the probe can be labeled during synthesis by incorporation of labelednucleotides. Alternatively, probe labeling can be done by nicktranslation or end-labeling. The label may comprise a fluorophore forfluorescence detection, or a ligand, such as biotin, which is detectedusing an enzyme-labeled binding molecule that binds to the ligand (e.g.,enzyme-labeled streptavidin) subsequent to hybridization.

In order to detect the presence of WSSV in a sample suspected ofcontaining WSSV, such as shrimp or other crustaceans, DNA is providedfrom the sample, as described above. The sample DNA is made available tocontact the probe before any hybridization of probe and target moleculecan occur. Thus, the DNA must be free from the cell and placed under theproper conditions before hybridization can occur. Additionally in someembodiments, it may be desirable to purify the DNA to eliminateproteins, lipids, and other cell components. A variety of methods ofnucleic acid purification, such as phenol-chloroform extraction, areknown to those skilled in the art (Maniatis, supra). Additionally, kitsare available from commercial sources for DNA extraction andpurification (e.g., IsoQuick® Nucleic Acid Extraction Kit (MicroProbeCorp., Bothell, Wash.); and QIAamp DNA Mini Kit (Qiagen, ValenciaCalif.)). Pre-hybridization purification is particularly useful forstandard filter hybridization assays.

In one embodiment, hybridization assays may be conducted directly oncell lysates, without the need to extract the nucleic acids. Thiseliminates several steps from the sample-handling process and speeds upthe assay. To perform such assays on crude cell lysates, a chaotropicagent is typically added to the cell lysates prepared as describedabove. The chaotropic agent stabilizes nucleic acids by inhibitingnuclease activity. Furthermore, the chaotropic agent allows sensitiveand stringent hybridization of short oligonucleotide probes to DNA atroom temperature (Van Ness and Chen, Nucl. Acids Res. 19:5143-5151(1991)). Suitable chaotropic agents include guanidinium chloride,guanidinium thiocyanate, sodium thiocyanate, lithium tetrachloroacetate,sodium perchlorate, rubidium tetrachloroacetate, potassium iodide, andcesium trifluoroacetate, among others. Typically, the chaotropic agentis present at a final concentration of about 3 M. If desired, one canadd formamide to the hybridization mixture, typically 30 to 50% byvolume.

Hybridization methods are well defined and include solution (i.e.,homogeneous) and solid phase (i.e., heterogeneous) hybridizationmethods. Typically, the sample DNA is probed (i.e. contacted underconditions which will permit nucleic acid hybridization) with a probederived from the WSSV diagnostic primer sequences disclosed herein. Thisinvolves contacting the probe and sample DNA in the presence of aninorganic or organic salt under the proper concentration and temperatureconditions. The probe and sample nucleic acids must be in contact for along enough time such that any possible hybridization between the probeand sample nucleic acid may occur. The concentration of probe or targetin the mixture will determine the time necessary for hybridization tooccur. The higher the probe or target concentration, the shorter thehybridization incubation time needed.

Various hybridization solutions can be employed. Typically, these maycomprise from about 20 to 60% by volume, preferably 30%, of a polarorganic solvent. A common hybridization solution employs about 30 to 50%by volume formamide, about 0.15 to 1 M sodium chloride, about 0.05 to0.1 M buffers, such as sodium citrate, Tris-HCl, PIPES or HEPES (pHrange about 6-9), about 0.05 to 0.2% detergent, such as sodiumdodecylsulfate (SDS), between 0.5 to 20 mM EDTA, FICOLL (AmershamBioscience Inc., Piscataway, N.J.) (molecular weight of about 300-500kilodaltons), polyvinylpyrrolidone (molecular weight of about 250-500kilodaltons), and serum albumin. Also included in a typicalhybridization solution may be unlabeled carrier nucleic acids from about0.1 to 5 mg/mL, fragmented nucleic DNA (e.g., calf thymus or salmonsperm DNA, or yeast RNA), and optionally from about 0.5 to 2% weight pervolume glycine. Other additives may also be included, such as volumeexclusion agents which include a variety of polar water-soluble orswellable agents (e.g., polyethylene glycol), anionic polymers (e.g.,polyacrylate or polymethylacrylate), and anionic saccharidic polymers(e.g., dextran sulfate).

Nucleic acid hybridization is adaptable to a variety of assay formats.One of the most suitable is the sandwich assay format. The sandwichassay is particularly adaptable to hybridization under non-denaturingconditions. A primary component of a sandwich-type assay is a solidsupport. The solid support has adsorbed to it or covalently coupled toit, an immobilized nucleic acid capture probe that is unlabeled and iscomplementary to one portion of the sample DNA sequence. Probesparticularly useful in the present invention are those derived from thepresent WSSV diagnostic sequences, as described above. The captured DNAis detected using a second probe that is labeled, as described above,and is complimentary to a different portion of the sample DNA sequence.The label may be detected using methods known in the art (e.g.,fluorescence, chemiluminescence, binding pair enzyme assay and thelike).

Hybridization methods may also be used in combination with nucleic acidamplification methods, such as PCR. For example, the instant WSSVdiagnostic sequences may be used as 3′ blocked detection probes ineither a homogeneous or heterogeneous assay format. For example, a probegenerated from the instant sequences may be 3′ blocked ornon-participatory and will not be extended by, or participate in, anucleic acid amplification reaction. Additionally, the probeincorporates a label that can serve as a reactive ligand that acts as apoint of attachment for the immobilization of the probe/analyte hybridor as a reporter to produce detectable signal. Accordingly, genomic DNAisolated from a sample suspected of harboring the WSSV is amplified bystandard primer-directed amplification protocols in the presence of anexcess of the 3′ blocked detection probe to produce amplificationproducts. Because the probe is 3′ blocked, it does not participate orinterfere with the amplification of the target. After the finalamplification cycle, the detection probe anneals to the relevant portionof the amplified DNA and the annealed complex is then captured on asupport through the reactive ligand.

The instant probe is versatile and may be designed in several alternateforms. The 3′ end of the probe may be blocked from participating in aprimer extension reaction by the attachment of a replication inhibitingmoiety. Typical replication inhibitor moieties include, but are notlimited to, dideoxynucleotides, 3′ deoxynucleotides, a sequence ofmismatched nucleosides or nucleotides, 3′ phosphate groups and chemicalagents, such as biotin, dinitrophenol, fluorescein, rhodamine, andcarbon chains. The replication inhibitor is covalently attached to the3′ hydroxy group of the 3′ terminal nucleotide of the non-participatoryprobe during chemical synthesis, using standard cyanoethylphosphoramidite chemistry. This process uses solid phase synthesischemistry in which the 3′ end is covalently attached to an insolublesupport (controlled pore glass, or “CPG”) while the newly synthesizedchain grows on the 5′ terminus. Within the context of the presentinvention, 3-deoxyribonucleotides are the preferred replicationinhibitors. Cordycepin (3-deoxyadenosine) is most preferred. Since thecordycepin will be attached to the 3′ terminal end of the probe, thesynthesis is initiated from a cordycepin covalently attached to CPG,5-dimethoxytrityl-N-benzoyl-3-deoxyadenosine (cordycepin),2-succinoyl-long chain alkylamino-CPG (Glen Research, Sterling, Va.).The dimethoxytrityl group is removed and the initiation of the chainsynthesis starts at the deprotected 5′ hydroxyl group of the solid phasecordycepin. After the synthesis is complete, the oligonucleotide probeis cleaved off the solid support leaving a free 2′ hydroxyl group on the3′-terminally attached cordycepin. Other reagents can also be attachedto the 3′ terminus during the synthesis of the non-participatory probeto serve as replication inhibitors. These include, but are not limitedto, other 3-deoxyribonucleotides, biotin, dinitrophenol, fluorescein,and digoxigenin. CPG supports, derivatized with each of these reagents,are available from commercial sources (e.g., Glen Research, Sterling,Va.; and CLONTECH Laboratories, Palo Alto, Calif.).

Alternatively, asymmetric amplification may be used to generate a strandcomplementary to the detection probe. Asymmetric PCR conditions forproducing single-stranded DNA are similar to the conditions describedabove for PCR; however, the primer concentrations are adjusted so thatone primer is in excess and the other primer is limiting. It iscontemplated that this procedure would increase the sensitivity of themethod. This improvement in sensitivity would occur by increasing thenumber of available single strands for binding with the detection probe.

Assessment of Infection Risk and DNA Damage or WSSV Inactivation

The methods for detecting the presence of and quantifying the amount ofWSSV in a sample disclosed herein may be used to assess the extent ofDNA damage or WSSV inactivation. For example, the methods disclosedherein may be used in combination with a chemical treatment to improvethe health and grow-out of shrimp. Specifically, during production andgrow-out, the shrimp, samples taken from the production facilities, orsamples taken from the shrimp's environment may be sampled and testedfor the presence of WSSV using the methods disclosed herein. If WSSV isfound, the facilities and/or the shrimp can be treated to kill orcontrol the virus. Because of the high sensitivity of the test, WSSV canbe detected early, before devastation and loss of the crop. Thus, use ofthe methods disclosed herein in combination with chemical interventioncan improve production efficiency and yield. Examples of chemicaltreatments include, but are not limited to, oxidative disinfectants suchas Virkon® S disinfectant (a registered trademark of E.I. Du Pont deNemours and Co.), peracetic acids, hydrogen peroxide, permanganate,potassium monopersulfate, hypochlorous acid, hypochlorite, iodine andthe like; probiotics, immunostimulants, feed supplements, andrecombinant protein/nucleic acids that prevent viral host binding. Afterthe chemical treatment, the shrimp can be sampled and retested todetermine if the treatment was successful in eradicating the virus.

In another embodiment, it is anticipated that the primers may be used invarious combinations to ascertain the integrity and extent of damage tothe viral genome resulting from chemical treatment. For example, theforward primer of WSSV54F (SEQ ID NO:3) and the reverse primer ofWSSV56R (SEQ ID NO:6) may be used in combination to produce anamplification product of 2008 bases. This longer product can form onlyif the viral genome remains intact and undamaged. Therefore, bycomparing the ratio of the smaller products (SEQ ID NO:10 or SEQ IDNO:11) to the longer product formed during amplification (or the absenceof the longer product), the extent of viral genome damage resulting fromchemical treatment or intervention can be assessed. This can aid inestablishing the efficacy of chemical treatment or intervention.

Detection Kits

In another embodiment, the invention provides a kit for the detection ofWSSV based on a nucleic acid amplification method. The kit comprises atleast one pair of WSSV diagnostic primer sequences, as described above.Additionally, the kit may further comprise at least one of the followingreagents: a thermostable DNA polymerase, a mixture of four differentdeoxynucleotide triphosphates, a nucleic acid-binding fluorescenceagent, at least one pair of internal sample control primers, at leastone internal template control and at least one pair of internal templatecontrol primers, a probe comprising a complementary sequence to aportion of at least one region of nucleic acid within the WSSV genomewhich is capable of being amplified with the WSSV diagnostic primersequences contained in the kit. The primers and other reagents of thekit may be in various forms, such as a liquid, dried, or tablet and maybe present in any suitable container or multiple containers, such asvials, tubes, and the like.

In another embodiment, the invention provides a kit for the detection ofWSSV based on a sandwich assay hybridization method. This kit comprisesa first component for the collection of samples from a shrimp or othercrustacean suspected of having contracted the WSSV and buffers for thedisbursement and lysis of the sample. A second component includes mediain either dry or liquid form for the hybridization of target and probenucleic acids, as well as for the removal of undesirable andnon-hybridized forms by washing. A third component includes a solidsupport (e.g., dipstick, bead, and the like) upon which is fixed (or towhich is conjugated) unlabeled nucleic acid probe(s) that is (are)derived from the isolated WSSV diagnostic primer sequences disclosedherein. A fourth component contains labeled probe that is complementaryto a second and different region of the same DNA strand to which theimmobilized, unlabeled nucleic acid probe of the third component ishybridized. The labeled probe may also be derived from the isolated WSSVdiagnostic primer sequences disclosed herein.

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various uses andconditions.

The meaning of abbreviations is as follows: “sec” means second(s), “min”means minute(s), “hr” means hour(s), “d” means day(s), “μL” meansmicroliter(s), “mL” means milliliter(s), “L” means liter(s), “μM” meansmicromolar, “mM” means millimolar, “nM” means nanomolar, “M” meansmolar, “mmol” means millimole(s), “μmol” mean micromole(s), “ng” meansnanogram(s), “fg” means femtogram(s), “μg” means microgram(s), “mg”means milligram(s), “g” means gram(s), “nm” means nanometer(s), “mU”means milliunit(s), “U” means unit(s), “r×n” means reaction(s), “PCR”means polymerase chain reaction, “OD” means optical density, “OD₂₆₀”means the optical density measured at a wavelength of 260 nm, “OD₂₈₀”means the optical density measured at a wavelength of 280 nm,“OD_(280/260)” means the ratio of the OD₂₈₀ value to the OD₂₆₀ value,“rpm” means revolutions per minute, “bp” means base pair(s), “CT” meansthe cycle number at which the buildup in fluorescence in the reactionexceeds the detection threshold, and “SPF” means certified specificpathogen free.

General Methods

Standard recombinant DNA and molecular cloning techniques used in theExamples are well known in the art and are described by Sambrook, J.,Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, byT. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with GeneFusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1984, and by Ausubel, F. M. et al., Current Protocols in MolecularBiology, Greene Publishing Assoc. and Wiley-Interscience, N.Y., 1987.

Analysis of genome sequences and primer designates was accomplishedusing the Vector NTI® Software Suite available from InforMax Inc.(Bethesda, Md.).

Enzymes and reagents used herein were purchased from the followingvendors:

-   -   Applied Biosystems, Foster City, Calif.: AmpliTaq (Catalog No.        N808-0160);    -   New England Biolabs, Beverly, Mass.: deoxynucleotide solution        mix (Catalog No. N0447S);    -   Sigma Genosys, The Woodlands, Tex.: Oligonucleotides;    -   Invitrogen Life Technologies, Carlsbad, Calif.: 4% Agarose        E-gels (Catalog No. G6018-02);    -   Qiagen, Valencia, Calif.: Proteinase K (Catalog No. 19131); and    -   RNase A, DNase-free (Catalog No. 19101).

Additionally, kits and reagents were purchased from the followingvendors: SYBR® Green PCR Master Mix (Applied Biosystems, Foster City,Calif.; Catalog No. 4309155); and QIAamp DNA Mini Kit (Qiagen, Valencia,Calif.; Catalog No. 51304).

All shrimp DNA samples were obtained from Donald V. Lightner, Departmentof Veterinary Science and Microbiology, The University of Arizona,Tucson, Ariz. 85721, USA. These included samples from certified diseasefree shrimp (SPF) and infected shrimp containing (Penaeus monodon-typebaculoviruses (MBV), Taura syndrome virus (TSV), white spot syndromevirus (WSSV), yellow head virus of P. monodon (YHV), InfectiousHypodermal and Hematopoietic Necrosis virus (IHHNV) and InfectiousMyonecrosis virus (IMNV).

Templates and Primers

DNA oligonucleotide sequences for synthesis of the synthetic WSSVtemplates were prepared from the White Spot Syndrome Virus (WSSV) DNAgenome (GenBank Accession Number AF332093; Yang, F., et al., J. Virology75 (23), 11811-11820 (2001)) and were synthesized using standardphosphoramidite chemistry or purchased commercially (Sigma GenosysCompany, The Woodlands, Tex.). The concentration and copy number of thesynthetic template targets were determined from spectrophotometricmeasurements at 260 nm (OD₂₆₀). The templates were diluted to specificcopy numbers in purified water and were used as the positive controlsand standards for assay quantification. Table 3 displays the genomelocations, sequence identification, and lengths of the template targets.The sequences of the primers useful for WSSV detection are given as SEQID NOs:1-8. TABLE 3 Template Sequences WSSV Genome Location TemplateSize (bp) SEQ ID NO: (GenBank AF332093) WSSV 77T 109 9 61335-61443 WSSV54T 128 10 31287-31414 WSSV 56T 148 11 33145-33292 WSSV 130T 125 12146110-146234

Examples 1-4 Demonstration of WSSV PCR Assay Using a Synthetic Target

The purpose of these Examples was to demonstrate the detection of theWSSV synthetic templates using PCR amplification with the primersdisclosed herein.

Template standards were prepared by 10 fold serial dilutions of thesynthetic WSSV templates (described above) in DNase free water.Generally, template concentrations of the standards ranged from 107 to 0copies/μL. A master mix was prepared by combining 25 μL/reaction of theSYBR® Green PCR Master Mix (Applied Biosystems, Foster City, Calif.;Catalog No. 4309155) with a volume of primer stock solutions (20 μM foreach of the WSSV primers) sufficient to give a final concentration of125 nM for each of the appropriate WSSV forward and reverse primers, asshown in Table 4, and enough DNase free water to make up a final volumeof 45 μL/reaction. The master mix was maintained on ice until use.

For each reaction, 5 μL of template standard was first added to the PCRreaction well and then 45 μL of the master mix was added. The reactionswere then thermal cycled for 40 cycles using a temperature program of95° C. for 15 sec and 60° C. for 1 min with an initial denaturing stepof 95° C. for 10 min. The amplifications were carried out in a MicroAmpoptical 96-well reaction plate using the ABI PRISM 7900 thermal cycler(Applied Biosystems, Foster City, Calif.). During each cycle, PCRproduct formation was detected by monitoring the increase influorescence arising from the interaction of the SYBR® Green reporterdye with the DNA amplification products. After completion of PCR, adissociation curve (also referred to herein as a melting curve) wasgenerated over the range of 60° C. to 95° C. Data were analyzed usingthe ABI PRISM 7900 SDS software. In addition, PCR product formation wasanalyzed by agarose gel electrophoresis using 4% agarose Egels(Invitrogen Life Technologies, Carlsbad, Calif.; Cat No. G6018-02) andthe gel manufacture's protocols.

The results, summarized in Table 4, demonstrate that the appropriatesize amplicon product was produced for each primer set when theappropriate WSSV template was present. The minimum detectable templatelevel was between 1 and 100 copies/r×n, depending on the primers used.Samples containing no template produced no detectable product.

Amplification (CT) and amplicon product formation were, respectively,inversely and directly proportional to the logarithm of the startingtemplate concentration. TABLE 4 Results of PCR Amplification using aSynthetic Target Forward Reverse Minimum Primer, Primer, TemplateProduct Detectable SEQ ID SEQ ID SEQ ID Size Template Example NO: NO:NO: (bp) (copies/rxn) 1 1 2 9 109 1 2 3 4 10 128 1 3 5 6 11 148 1 4 7 812 125 100

Examples 5-8 Detection and Quantification of WSSV DNA from InfectedShrimp Tissue Using a PCR Assay

The purpose of these Examples was to demonstrate the detection andquantification of WSSV in infected shrimp using a PCR assay with theprimers disclosed herein.

In these Examples, serial dilutions of the appropriate synthetictemplate DNA (described above) ranging from 10⁶ to 10⁰ copies perreaction, were amplified using the conditions stated in Examples 1-4. Astandard curve (not shown) was generated using the CT values determinedfrom each of the synthetic template concentrations by plotting the CTvalues, with 95% confidence intervals, against the logarithm of theinitial template copy numbers in the standards. The slope of this curve(i.e., CT versus log concentration) was then used to estimate the copiesof viral genome in an unknown sample from their respective CT values.

Genomic DNA from shrimp infected with a China strain of the WSSV wasused. The total DNA (230 ng/μL) isolated from the infected shrimp gillswas serially diluted in purified water and used to provide a series ofsamples ranging in DNA concentration from 1 ng/μL to 1 fg/μL of totalDNA. Negative controls included a water control containing no templateand two DNA shrimp samples (10 ng/r×n) obtained by extracting DNA fromtwo strains of non-infected (SPF) shrimp (Litopenaeus vannamei andPenaeus monodon).

The diluted samples were then amplified using one of the primer pairs(see Table 5) and the same amplification, master mix, thermal cyclingconditions and instrument stated in Examples 1-4. The CT value for eachdiluted DNA sample was then assessed from the PCR amplificationreactions. The copies of viral genome in the samples were then estimatedfrom the CT value and the slope of the standard CT versus log templateconcentration plot. The PCR products were also analyzed by agarose gelelectrophoresis, as described in Examples 1-4.

The results are summarized in Table 5. In the table, the WSSV copynumber per reaction is given as the mean of three replicates along withthe 95% confidence interval. The results indicate that all of the primersets produced the correct amplicon product size from the infected shrimpDNA and detected WSSV DNA in the infected shrimp samples. The detectionlimits ranged from about 2 copies/r×n to about 300 copies/r×n of theviral genome, depending on the primer pair used. No amplificationproducts were detected in the water control sample or the two SPF shrimpsamples. The results obtained with the negative control samplesdemonstrate that the assay is non-responsive to non-viral DNA from thetwo shrimp strains tested. TABLE 5 Results of Detection of WSSV DNA fromInfected Shrimp Tissue Forward Reverse Infected Ex- primer primer Shrimpam- SEQ ID SEQ ID DNA/rxn WSSV ple NO: NO: (ng) CT copies/rxn 5 1 2 122.9      48 ± 1 × 10⁴ 5 1 2 0.1 26.5     3.9 ± 0.1 × 10⁴   5 1 2 0.0129.8     3.0 ± 0.2 × 10³   5 1 2 0.001 33.2     3.5 ± 0.4 × 10²   5 1 20.0001 37.4     1.7 ± 0.3 × 10¹   5 1 2 0.00001 >40 0 5 1 2 0.000001 >400 5 1 2 0 >40 0 (water) 5 1 2 0 38.9 0 (SPF L. vannamei (10 ng)) 5 1 2 037.9 0 (SPF P. monodon (10 ng)) 6 3 4 1 19.9      18 ± 3 × 10⁴ 6 3 4 0.123.6     1.7 ± 0.1 × 10⁴   6 3 4 0.01 27.1     1.7 ± 0.1 × 10³   6 3 40.001 30.9     1.5 ± 0.1 × 10²   6 3 4 0.0001 35.5   7 ± 1 6 3 4 0.0000136.5   2 ± 3 6 3 4 0.000001 >40 0 6 3 4 0 >40 0 (water) 6 3 4 0 >40 0(SPF L. vannamei (10 ng)) 6 3 4 0 >40 0 (SPF P. monodon (10 ng)) 7 5 6 119.0      36 ± 2 × 10⁴ 7 5 6 0.1 33.7       3.4 ± 0.01 × 10⁴ 7 5 6 0.0130.5     2.5 ± 0.4 × 10³   7 5 6 0.001 26.1     1.2 ± 0.6 × 10²   7 5 60.0001 22.4   13 ± 8  7 5 6 0.00001 36.1   2 ± 1 7 5 6 0.000001 >40 0 75 6 0 >40 0 (water) 7 5 6 0 >40 0 (SPF L. vannamei (10 ng)) 7 5 6 0 >400 (SPF P. monodon (10 ng)) 8 7 8 1 23.3      88 ± 2 × 10⁴ 8 7 8 0.1 28.3    4 ± 3 × 10⁴   8 7 8 0.01 31.9     4.3 ± 0.6 × 10³   8 7 8 0.001 36.3    3 ± 1 × 10²   8 7 8 0.0001 >40 0 8 7 8 0.00001 >40 0 8 7 80.000001 >40 0 8 7 8 0 >40 0 (water) 8 7 8 0 >40 0 (SPF L. vannamei (10ng)) 8 7 8 0 >40 0 (SPF P. monodon (10 ng))

Example 9 Specificity of WSSV Primers

The purpose of this Example was to demonstrate that the primersdisclosed herein amplify DNA from WSSV strains from differentgeographical areas of the World, but do not amplify DNA of shrimpinfected with other shrimp pathogens.

In this Example, DNA from shrimp infected with other shrimp pathogenswas used. Specifically, DNA samples isolated from shrimp infected withWSSV strains from different geographical regions (i.e., Hawaii,Philippines, Thailand, Panama, Mexico Mozambique and Madagascar) alongwith non-WSSV shrimp viruses (MBV, IHHNV, YHV and IMNV) were testedusing the primers and PCR method described in Examples 5-8.

All WSSV strains were detected with similar detection limits as thestrain described in Examples 5-8. No PCR amplification was observed whentesting the non-WSSV infected shrimp DNA samples. These findings takentogether demonstrate that the WSSV diagnostic primer sequences andmethods disclosed herein are selective for WSSV and that the primers donot react with shrimp DNA or other shrimp viruses.

Example 10 Detection of WSSV DNA in Combination with an Internal SampleControl Using PCR

The purpose of this Example was to demonstrate that the WSSV primersdisclosed herein can be used in combination with internal sample control(ISC) primers to produce an ISC product in addition to the WSSV product.The results presented below demonstrate that the ISC primersindependently amplify sample DNA and do not interfere with theamplification of WSSV DNA. The presence of the ISC product provides amarker that can be used as an indication that sample DNA of sufficientquantity and quality had been recovered for testing.

ISC primers were derived from the Penaeus monodon actin 1 gene sequence(GenBank: AF100986). In order to promote preferential amplification ofthe WSSV amplicon, the ISC primers were designed to amplify a DNAfragment that was larger than the target WSSV amplicons. The ISC primerpair sequences are given as SEQ ID NOs:13,14, and SEQ ID NOs:15,16 (seeTable 2).

Samples containing WSSV and shrimp actin DNA were prepared by 10-foldserial dilutions of a genomic DNA preparation from a WSSV infectedshrimp (Penaeus monodon) with DNase free water. The DNA content of thesamples ranged from 0.1 ng to 0.1 pg per reaction. Genomic DNA (10 ng)from a non-infected shrimp was then added to each WSSV sample and tonegative control samples containing no WSSV DNA.

A master PCR mix was prepared by combining 15 μL/reaction of the SYBR®Green PCR Master Mix (Applied Biosystems, Foster City, Calif.; CatalogNo. 4309155) with a volume of primer stock solutions (20 μM for each ofthe WSSV54 primers and 10 μM for each of the actin primers) sufficientto give a final concentration of 125 nM for each of the WSSV54 forwardand reverse primers (SEQ ID NOs:3 and 4, respectively) and 32 nM foreach of the actin forward and reverse primers (SEQ ID NOs:13 and 14 orSEQ ID NOs:15 and 16, respectively). DNase free water was added to makeup a final volume of 25 μL/reaction. The master mix was maintained onice until use.

For each reaction, 5 μL of the samples was first added to the PCRreaction wells and then 25 μL of the master mix was added. The reactionswere then thermal cycled for 40 cycles using a temperature program of95° C. for 15 sec and 60° C. for 1 min with an initial denaturing stepof 95° C. for 5 min. Amplification was carried out in a MicroAmp optical96-well reaction plate using the ABI PRISM 7900 thermal cycler (AppliedBiosystems, Foster City, Calif.).

During each cycle, product formation was monitored by the CT valuedetermined from the increase in fluorescence arising from theinteraction of the SYBR® Green reporter dye with the DNA amplificationproducts, as described above. After 40 cycles a dissociation curve(melting curve) was generated over the range of 60° C. to 95° C. Datawere analyzed using the ABI PRISM 7900 SDS software. In addition, PCRproduct formation was analyzed by agarose gel electrophoresis using 4%agarose Egels (Invitrogen Life Technologies, Carlsbad, Calif.; Cat No.G6018-02) and the gel manufacture's protocols. The results obtainedusing ISC primers ActinF2 (SEQ ID NO:13) and ActinR2 (SEQ ID NO:14) areshown in FIGS. 1A and 1B, which demonstrate the simultaneousamplification of both template targets. The specific WSSV DNA produced a128 bp product with a melting temperature of 78.5° C. The actin ISCproduced a 239 bp product (Tm=83.8° C.). The WSSV product and actininternal control products were detected by both melting curve analysis(FIG. 1A) and gel electrophoresis (FIG. 1B) based on these size andmelting temperature differences. In the absence of WSSV target and atvarious WSSV target concentrations, formation of the ISC product wasdetected as a single melting-temperature peak at 83.8° C. (as shown inFIG. 1A) and by electrophoresis (as shown in FIG. 1B). In all samplescontaining the WSSV template, the specific WSSV amplicon was detected byboth melting temperature (Tm=78.5° C.) and by gel electrophoresis. Theseresults demonstrate that the actin ISC template co-amplifies with theWSSV template and that the PCR amplification and limit of detection ofthe PCR assay (0.1 pg WSSV DNA) are unaffected by the presence of theISC.

Examples 11 and 12 Real Time Detection of WSSV DNA Using FluorescentlyLabeled Probes

This Example demonstrates that the WSSV primers disclosed herein can beused in combination with fluorescently labeled probes for real timedetection and quantification of WSSV.

Gene sequences for construction of the fluorescently labeled probes wereselected by analysis of the WSSV genes and test amplicons using PrimerExpress® v2.0 software, purchased from Applied BioSystems Inc. (FosterCity, Calif. 94404). The probe sequences were chosen to fall within theproximal ends of the specific WSSV test amplicons and were 50 to 110bases in length, depending on the size and sequence of the amplicon.Preference for the probe sequences was given to regions with G/C contentof 30 to 80% and with higher C than G content, and with no 5′ G.Generally, probe sequences were selected having a Tm of 8 to 10° C.above the respective Tm of the test primers. Probes sequences whichcross-hybridized to other species were not selected for use. The probesequences selected to meet these criteria are listed in Table 6.

For real-time detection, the probe sequences were dual labeled. Twodifferent labeling approaches were employed. The 5′ end of the probeswere labeled with a fluorophore (6FAM™, Applied Biosystems). The 3′ endwas labeled either with a quencher dye or in the case of minor grovebinding (MGB) probe, the 3′ end was labeled with a quencher dye and aminor grove binder complex. The labeled probes were prepared andpurchased commercially from Applied BioSystems. TABLE 6 FlourescentlyLabeled Probe Sequences SEQ ID GenBank 5′ 3′ Probe NO: No: LocationLabel Label(s) WSSV 54PM 17 AF332093 31345-31364 FAM¹ MGB² WSSV 54PT 18AF332093 31345-31364 FAM TAMRA³ WSSV 77PT 19 AF332093 61363-61385 FAMTAMRA¹FAM is 6FAM ™ reagent, Applied Biosystems²MGB is MGB ™ Applied Biosystems³TAMRA is 6-carboxytetramethylrhodamine

Template standards were prepared by 10-fold serial dilutions of thesynthetic WSSV templates (described above) in DNase free water.Generally, template concentrations of the standards ranged from 107 to 0copies/μL. A master mix was prepared by combining 25 μL/reaction of theTaqMan® Universal Master Mix (Applied Biosystems, Foster City, Calif.;Catalog No. 4326708) with a volume of primer stock solutions (20 μM foreach of the WSSV primers) sufficient to give a final concentration of125 nM for each of the appropriate WSSV forward and reverse primers, asshown in Table 7, a volume of probe stock solution to give a finalconcentration of 50 nM and enough DNase free water to make up a finalvolume of 45 μL/reaction. The master mix was maintained on ice untiluse.

For each reaction, 5 μL of template standard and then 45 μL of themaster mix were added to each PCR reaction well. The reactions were thenthermal cycled for 40 cycles using a temperature program of 95° C. for15 sec and 60° C. for 1 min with an initial denaturing step of 95° C.for 10 min. The amplifications were carried out in a MicroAmp optical96-well reaction plate using the ABI PRISM 7900 thermal cycler (AppliedBiosystems, Foster City, Calif.). During each cycle, PCR productformation was detected by monitoring the increase in fluorescencearising from the fluorescently labeled probe.

Data were analyzed using ABI SDS 2.2 software. In addition, PCR productformation was analyzed by agarose gel electrophoresis using 4% agaroseEgels (Invitrogen Life Technologies, Carlsbad, Calif.; Cat No. G6018-02)and the manufacture's protocols.

The results, summarized in Table 7, demonstrate that the appropriatesize amplicon product was produced for each primer/probe set when theappropriate WSSV template was present. The minimum detectable templatelevel was between 100 and 1000 copies/r×n, depending on the primers andprobe used. Samples containing no template produced no detectableproduct.

Amplification (CT) and amplicon product formation were, respectively,inversely and directly proportional to the logarithm of the startingtemplate concentration. TABLE 7 Results of PCR Amplification Using aSynthetic Target Forward Reverse Minimum Primer, Primer, Template ProbeProduct Detectable Exam- SEQ ID SEQ ID SEQ ID SEQ ID Size Template pleNO: NO: NO: NO: (bp) (copies/rxn) 11 3 4 10 17 128 1000 12 1 2 9 19 109100

1. An isolated WSSV diagnostic primer sequence as set forth in SEQ IDNO:1 or an isolated nucleic acid molecule that is completelycomplementary to SEQ ID NO:1.
 2. An isolated WSSV diagnostic primersequence as set forth in SEQ ID NO:2 or an isolated nucleic acidmolecule that is completely complementary to SEQ ID NO:2.
 3. An isolatedWSSV diagnostic primer sequence as set forth in SEQ ID NO:3 or anisolated nucleic acid molecule that is completely complementary to SEQID NO:3.
 4. An isolated WSSV diagnostic primer sequence as set forth inSEQ ID NO:4 or an isolated nucleic acid molecule that is completelycomplementary to SEQ ID NO:4.
 5. An isolated WSSV diagnostic primersequence as set forth in SEQ ID NO:5 or an isolated nucleic acidmolecule that is completely complementary to SEQ ID NO:5.
 6. An isolatedWSSV diagnostic primer sequence as set forth in SEQ ID NO:6 or anisolated nucleic acid molecule that is completely complementary to SEQID NO:6.
 7. An isolated WSSV diagnostic primer sequence as set forth inSEQ ID NO:7 or an isolated nucleic acid molecule that is completelycomplementary to SEQ ID NO:7.
 8. An isolated WSSV diagnostic primersequence as set forth in SEQ ID NO:8 or an isolated nucleic acidmolecule that is completely complementary to SEQ ID NO:8.
 9. A pair oftwo different WSSV diagnostic primer sequences of any of claims 1-8wherein the pair is capable of priming a nucleic acid amplificationreaction that amplifies a region of nucleic acid within the WSSV genome.10. A pair of two different WSSV diagnostic primer sequences accordingto claim 9 wherein the pair is selected from the group consisting of SEQID NOs:1 and 2, SEQ ID NOs:3 and 4, SEQ ID NOs:5 and 6, SEQ ID NOs:7 and8, SEQ ID NO:3 and 6, SEQ ID NO:3 and the complete compliment of SEQ IDNO:5, and SEQ ID NO:6 and the complete compliment of SEQ ID NO:4.
 11. Akit for the detection of WSSV comprising at least one pair of WSSVdiagnostic primer sequences of claim
 9. 12. A kit for the detection ofWSSV according to claim 11 wherein the kit further comprises at leastone reagent selected from the group consisting of a thermostablepolymerase, a mixture of four different deoxynucleotide triphosphates, anucleic acid-binding fluorescent molecule, at least one pair of internalsample control primers, at least one internal template control and atleast one pair of internal template control primers, and a probecomprising a complementary sequence to a portion of at least one regionof nucleic acid within the WSSV genome which is capable of beingamplified with the at least one pair of WSSV diagnostic primersequences.
 13. A method for detecting the presence of WSSV in a samplecomprising: (i) providing DNA from a sample suspected of containing theWSSV; and (ii) probing the DNA with a probe derived from the isolatedWSSV diagnostic primer sequence of any of claims 1-8 under suitablehybridization conditions; wherein the identification of a hybridizablenucleic acid fragment confirms the presence of WSSV.
 14. A method fordetecting the presence of WSSV in a sample according to claim 13 whereinthe probe derived from the isolated WSSV diagnostic primer sequence isselected from the group consisting of SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, and the complete complimentary sequences thereof.
 15. Amethod according to claim 13 wherein the probe contains a replicationinhibiting moiety at the 3′ end.
 16. A method according to claim 15wherein the replication inhibiting moiety is selected from the groupconsisting of dideoxynucleotides, 3′ deoxynucleotides, a sequence ofmismatched nucleosides or nucleotides, 3′ phosphate groups and chemicalagents.
 17. A method according to claim 16 where in the 3′deoxynucleotide is cordycepin.
 18. A method for detecting the presenceof WSSV in a sample comprising: (i) providing DNA from a samplesuspected of containing WSSV; and (ii) amplifying the DNA with at leastone pair of WSSV diagnostic primer sequences of claim 9 such thatamplification products are generated; wherein the presence ofamplification products confirms the presence of WSSV.
 19. A method fordetecting the presence of WSSV in a sample according to claim 18 whereinthe amplifying of (ii) is done using the polymerase chain reaction. 20.A method for detecting the presence of WSSV in a sample according toclaim 18 wherein the amplifying of (ii) is done in the presence of anucleic acid-binding fluorescent agent or a fluorescently labeled probeand the presence of amplification products is confirmed usingfluorescence detection.
 21. A method according to claim 20 wherein thefluorescently labeled probe is selected from the group consisting of SEQID NO:17, 18, and
 19. 22. A method according to claim 18 wherein atleast one pair of internal sample control primers is included in theamplifying of (ii) to produce an internal sample control product.
 23. Amethod according to claim 22 wherein the at least one pair of internalsample control primers is selected from the group consisting of SEQ IDNOs:13, 14 and SEQ ID NOs:15,16.
 24. A method according to claim 18wherein at least one pair of internal template control primers and atleast one internal template control are included in the amplifying of(ii) to produce an internal template control product.
 25. A method forquantifying the amount of WSSV in a sample comprising: (i) providing DNAfrom a sample suspected of containing WSSV; (ii) amplifying the DNA withat least one pair of WSSV diagnostic primer sequences of claim 9 bythermal cycling between at least a denaturing temperature and anextension temperature in the presence of a nucleic acid-bindingfluorescent agent or a fluorescently labeled probe; (iii) measuring theamount of fluorescence generated by the nucleic acid-binding fluorescentagent or the fluorescently labeled probe during the thermal cycling;(iv) determining a cycle threshold number at which the amount offluorescence generated by the nucleic acid-binding fluorescent agent orthe fluorescently labeled probe reaches a fixed threshold value above abaseline value; and (v) calculating the amount of WSSV in the sample bycomparing the cycle threshold number determined for the WSSV in thesample with a standard curve of the cycle threshold number versus thelogarithm of template concentration determined using standard solutionsof known concentration.
 26. A method according to claim 25 where in thefluorescently labeled probe is selected from the group consisting of SEQID NO:17, 18, and
 19. 27. A method according to any of claims 13, 18, or25 wherein the method is used to assess DNA damage or WSSV inactivation.28. A method according to any of claims 13, 18, or 25 wherein the methodis used in combination with chemical treatment to improve the health andgrow-out of shrimp.